Lipases play an important role in asymmetric biocatalysis. Their broad substrate specificity, generally high regio- and enantio-selectivity, as well as their ability to function in aqueous and organic reaction medium make them versatile tools for the kinetic resolution, derivatization, chiral synthesis, and polymerization of esters. Lipases can catalyze the formation, hydrolysis, and substitution (transesterification) of ester bonds, amid bonds, and the like. They are important biocatalysts in production of chiral building blocks for fine chemicals and pharmaceuticals, as well as in bulk products such as laundry detergent.
Suitable enzymes for a particular substrate can be identified by screening natural lipases or can be tailored by protein engineering. In the latter case, rational protein design, random mutagenesis, and DNA shuffling have generated laboratory catalysts with altered specificity, selectivity, and stability. However, very few natural and lab-made lipases show activity and enantioselectivity for bulky substrates such as esters of large secondary and tertiary alcohols. It has been hypothesized that the cause for the poor turnover of these substrates arises from steric constraints in the lipase active site, yet protein engineers have so far failed to generate improved biocatalysts. Tailoring these enzymes to novel, unnatural substrates is one of the primary challenges of protein engineering. Circular permutation may provide the ability to meet such challenges.
Circular permutation is a technique where the normal termini of a polypeptide are linked and new termini are created by breaking the backbone elsewhere. In many polypeptides, the normal termini are in close proximity and can be joined by a short amino acid sequence. The break in the polypeptide backbone can be at any point, preferably at a point where the function and folding of the polypeptide are not destroyed. Circular permutation creates new C- and N-termini, so the technique is often used in the creation of fusion proteins where the fused peptide or protein is attached at a different place on the host protein. For example, if the natural termini are at the interior of the base protein, it may be disruptive to attach a peptide or protein at the natural termini. By changing the attachment location to a place near the exterior of the host protein, stability of the host protein may be maintained.
Circular permutation provides an experimental way to investigate the biophysical consequences of backbone rearrangement or removal on ligand binding in ways not available using traditional deletion mutants. Circularly permuted proteins have been used previously to investigate the protein folding problem (Yang Y, et al. (1993) Proc Natl Acad Sci US. 90:11980-1984; Graf R, et al. (1996) Proc Natl Acad Sci USA 93:11591-11596). Both naturally occurring and synthetic circularly permuted proteins have been identified (Heinemann U, et al. (1995) Prog Biophys Molec Biol 64:122-143; Lindqvist Y, et al. (1997) Curr Opinion Struc Biol 7:422-427; Goldenberg D P, et al. (1983) J Mol Biol 164:407-413; Luger K, et al. (1989) Science 243-206-209). U.S. Pat. No. 5,635,599 to Pastan et al. discloses fusion proteins created from circularly permuted interleukin 4 (IL4).
As mentioned above, circular permutants generally are created by disrupting the polypeptide chain at a selected point to create new termini and bridging the two natural termini either directly or through a linker such as an amino acid linker. Circular permutation thus has the effect of essentially preserving the sequence and identity of the amino acids of a protein, while generating new termini at different locations. Moreover, the tertiary structure of the protein is generally conserved. Circularly permuted proteins can be made chemically or created by recombinant techniques.